Transcriptome-wide analysis of wild Asari (=Manila) clams affected by the Brown Muscle Disease: etiology and impacts of the disease

Recently, we reported an emerging pathology named Brown Muscle Disease (BMD) affecting Asari clams inhabiting the most productive area for this species in France, the Arcachon Bay. The main macroscopic feature of the pathology relies on the atrophy of the posterior adductor muscle, affecting the ability of clams to burry. The research of the etiological agent of BMD privileged a viral infection. Contrary to healthy clams, infected animals are always found at the surface of the sediment and exhibit 30 nm virus-like particles in muscle, granulocytic and rectal cells. In order to get more insights on the etiology and impacts of the BMD on clams, we took advantage in the present study of next generation sequencing technologies. An RNA-Seq approach was used (i) to test whether viral RNA sequences can be specifically found in the transcriptome of diseased animals and (ii) to identify the genes that are differentially regulated between diseased and healthy clams. Contrary to healthy buried animals, in diseased clams one sequence showing extensive homologies with retroviridae-related genes was detected. Among the biological processes that were affected in diseased clams, the synaptic transmission process was the most represented. To deepen this result, a new sampling was carried out and the transcription level of genes involved in synaptic transmission was determined in healthy and diseased clams but also in clams with no visible sign of pathology but located at the surface of the sediment. Our findings suggest that muscle atrophy is a latter sign of the pathology and that nervous system could be instead a primary target of the BMD agent. Two pools of total RNA material were formed using a total of 5 clams : a pool of 3 animals exhibiting signs BMD (SURF+) and a pool of 2 animals with no sign of BMD (BUR-). Pools were done with equal amount of RNA from each samples and sequenced on one flow cell.

近期，本团队报道了一种新兴病害——褐肌病（Brown Muscle Disease, BMD），该病害侵染栖息于法国阿卡雄湾（Arcachon Bay）该物种核心养殖区的浅蜊（Asari clams）。该病害的主要宏观特征为后闭壳肌萎缩，进而影响浅蜊的掘穴能力。针对BMD病原的初步研究指向病毒感染。与健康浅蜊不同，染病个体始终暴露于沉积物表层，且在其肌肉、颗粒细胞及直肠细胞中可观察到30 nm大小的病毒样颗粒。为深入解析BMD的病原学特征及其对浅蜊的影响，本研究借助下一代测序（next generation sequencing, NGS）技术开展相关实验。本研究采用RNA测序（RNA-Seq）技术，旨在达成两个目标：其一，验证染病个体转录组中是否特异性存在病毒RNA序列；其二，筛选染病与健康浅蜊间差异表达的基因。与正常掘穴的健康个体不同，染病浅蜊中检测到一段与逆转录病毒科（Retroviridae）相关基因具有高度同源性的序列。在染病个体受影响的生物学过程中，突触传递过程的富集程度最高。为进一步验证该结果，本研究开展了新一轮采样，并对健康浅蜊、染病浅蜊，以及无可见病害症状但同样暴露于沉积物表层的浅蜊体内突触传递相关基因的转录水平进行了定量检测。本研究结果表明，肌肉萎缩是该病害的晚期症状，而神经系统可能才是BMD病原的主要侵染靶点。本研究共收集5只浅蜊，分为两个总RNA混合池：其一为3只表现出BMD症状的暴露个体（SURF+），其二为2只无BMD症状的掘穴个体（BUR-）。混合池按等质量RNA比例制备，随后在单个测序流动槽（flow cell）上完成测序。